WO2018124766A2 - 키메라 항원 수용체 및 이를 발현하는 자연 살해 세포 - Google Patents

키메라 항원 수용체 및 이를 발현하는 자연 살해 세포 Download PDF

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WO2018124766A2
WO2018124766A2 PCT/KR2017/015635 KR2017015635W WO2018124766A2 WO 2018124766 A2 WO2018124766 A2 WO 2018124766A2 KR 2017015635 W KR2017015635 W KR 2017015635W WO 2018124766 A2 WO2018124766 A2 WO 2018124766A2
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car
domain
chimeric antigen
intracellular signaling
nucleotides
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PCT/KR2017/015635
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English (en)
French (fr)
Korean (ko)
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WO2018124766A9 (ko
WO2018124766A3 (ko
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황유경
조성유
원성용
임호용
허정현
정미영
김현아
권수현
이은솔
김한솔
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주식회사 녹십자랩셀
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Priority to JP2019536074A priority Critical patent/JP6971319B2/ja
Priority to EP17886739.6A priority patent/EP3567049A4/en
Priority to CA3061898A priority patent/CA3061898A1/en
Priority to CN201780081622.3A priority patent/CN110121505B/zh
Priority to US16/474,426 priority patent/US20190336533A1/en
Priority to AU2017384900A priority patent/AU2017384900B2/en
Publication of WO2018124766A2 publication Critical patent/WO2018124766A2/ko
Publication of WO2018124766A3 publication Critical patent/WO2018124766A3/ko
Publication of WO2018124766A9 publication Critical patent/WO2018124766A9/ko
Priority to US17/845,793 priority patent/US20230025506A1/en

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Definitions

  • the present invention relates to chimeric antigen receptors and natural killer cells expressing the same.
  • NK cells Human natural killer cells play an important role in the innate immune defense against malignant cells, making them suitable for adaptive immune therapy and adoptive cellular immunotherapy. can do.
  • NK cells Human natural killer cells
  • Chimeric antigen receptors for autoimmune cell therapy have been developed. Chimeric antigen receptors are known to be able to reprogram T cells to activate them to enhance the therapeutic effect on certain cancers or to help overcome cancer cell resistance to treatment.
  • OX40 ligand (CD252) is a protein belonging to the TNFR superfamily, which is known to be expressed in antigen-presenting cells (APCs), some natural killer cells and some B cells, and several hours after activation of these cells. It is known to be expressed in a few days.
  • OX40 (CD134), a receptor for OX40 ligand (CD252), is known to be expressed in T cells, and is known to be expressed after 24 hours when T cells are activated by antigen and CD28. Expression of CD134 is known to further enhance T cell responses by CD28 activation, thereby increasing T cell proliferation, cytokine secretion and survival.
  • OX40 ligand in enhancing anticancer activity of natural killer cells is not well known, and no attempt has been made to use OX40 ligand for chimeric antigen receptors.
  • CD252 In vitro, lymphoma cell lines were used to detect their anti-tumor responses in the presence of rituximab specific for CD20 molecules. Investigate.
  • NKG2D is a major receptor for NK cell activation and binds to MHC class 1 chain-related A (MICA), MICB, and various UL-16-binding proteins (ULBP) that are preferentially expressed after cellular stress, infection or cytotoxicity.
  • MICA MHC class 1 chain-related A
  • MICB MHC class 1 chain-related A
  • ULBP UL-16-binding proteins
  • Chimeric receptors containing NKG2D and various signal molecules known to cause cytotoxicity in NK cells were constructed and expressed in NK92MI cells. Since then, their anti-tumor effects were evaluated in vitro.
  • the present inventors may use a chimeric antigen receptor including an OX40 ligand as a way to overcome the limitation of autoimmune cell treatment using natural killer cells, and use the OX40 ligand in comparison with a known signaling domain.
  • the chimeric antigen receptor was found to be remarkably excellent in the antitumor activity enhancing effect of natural killer cells to complete the present invention.
  • An object of the present invention is to provide a chimeric antigen receptor for enhancing the anticancer immune cell treatment efficiency using natural killer cells.
  • a chimeric antigen receptor comprising an intracellular signaling domain comprising all or a portion of an OX40 ligand (CD252).
  • transmembrane domain is linked to the intracellular signaling domain; A spacer domain linked to the transmembrane domain; And an extracellular domain linked to said spacer domain.
  • extracellular domain of claim 2 wherein the extracellular domain comprises at least one selected from the group consisting of an antigen-binding fragment of an antibody, an Fc receptor, a natural cytotoxicity receptor, NKG2D, 2B4, and DNAM-1. It comprises, chimeric antigen receptor.
  • Fv fragment is a single-chain variable fragment (Single-chain variable fragment, ScFv), chimeric antigen receptor.
  • chimeric antigen receptor according to the above 5, wherein the natural cytotoxicity receptor is selected from the group consisting of NKp46, NKp30, NKp44, NKp80 and NKp65 receptors.
  • transmembrane domain includes all or part of any one selected from the group consisting of CD8 ⁇ and CD28, chimeric antigen receptor.
  • the intracellular signaling domain additionally comprises all or part of CD3 ⁇ (CD3-zeta), chimeric antigen receptor.
  • Chimeric antigen receptor comprising the following intracellular signaling domains: a first intracellular signaling domain comprising all or part of any one selected from the group consisting of CD28 and 4-1BB; A second intracellular signaling domain comprising all or a portion of any one selected from the group consisting of an OX40 ligand, OX40 and 4-1BB; And a third intracellular signaling domain comprising all or a portion of CD3 ⁇ (wherein the first, second and third intracellular signaling domains are located in the intracellular direction in the cell membrane in order).
  • transmembrane domain according to 15 above, which is linked to the intracellular signaling domain; A spacer domain linked to the transmembrane domain; And an extracellular domain linked to said spacer domain.
  • the chimeric antigen receptor according to the above 16 further comprising a signal sequence linked to the extracellular domain.
  • transmembrane domain comprises all or part of any one selected from the group consisting of CD8 ⁇ and CD28.
  • first intracellular signaling domain comprises all or part of CD28;
  • second intracellular signaling domain comprises all or a portion of an OX40 ligand;
  • third intracellular signaling domain comprises all or part of CD3 ⁇ , chimeric antigen receptor.
  • NK cell natural killer cell
  • a pharmaceutical composition for treating tumors comprising the immune cells of the above 23 as an active ingredient.
  • composition for treating tumors according to 25 above further comprising an antibody as an active ingredient when the extracellular domain is an Fc receptor.
  • nucleic acid sequence is SEQ ID NO: 33, 41, 43, 45, 47, 49, 51, 53, 55, 69, 71, 77, 81, 83, 85, 87, 89, 91, and 93
  • nucleic acid sequence is SEQ ID NO: 32, 40, 42, 44, 46, 48, 50, 52, 54, 68, 70, 76, 80, 82, 84, 86, 88, 90 and 92
  • a nucleic acid sequence comprising at least one nucleic acid sequence selected from the group consisting of or variants thereof having at least 80% sequence identity.
  • a method of treating tumor comprising administering the above immune cells to a subject.
  • the chimeric antigen receptor according to the present invention is excellent in natural killer cell activation efficiency.
  • the chimeric antigen receptor according to the invention can be used with various cancer target antibodies depending on the type of cancer targeted.
  • the chimeric antigen receptor according to the invention can be applied to a variety of carcinomas by applying various antigen recognition sites.
  • Natural killer cells expressing the chimeric antigen receptor according to the present invention have excellent cytotoxicity against cancer cells.
  • Natural killer cells expressing the chimeric antigen receptor according to the present invention can be usefully used for the treatment of immune cells.
  • CD16V-Z CAR (1st generation), CD16V-28Z CAR (2nd generation), CD16V-BBZ CAR (2nd generation), CD16V-OX40Z CAR (2nd generation), or CD16V-28OX40LZ according to an embodiment of the invention.
  • the expression level of each CAR in NK92MI cells transduced with CAR (3rd generation) is shown.
  • CD16V-Z CAR (1st generation), CD16V-28Z CAR (2nd generation), CD16V-BBZ CAR (2nd generation), CD16V-OX40Z CAR (2nd generation), or CD16V-28OX40LZ according to an embodiment of the invention.
  • the intrinsic cell killing ability of NK92MI cells transduced with CAR (3rd generation) is shown through cytotoxicity assessment for K562 cells.
  • 1C shows a CD16V-Z CAR (1st generation), CD16V-28Z CAR (2nd generation), CD16V-BBZ CAR (2nd generation), CD16V-OX40Z CAR (2nd generation), or CD16V-28OX40LZ according to an embodiment of the invention. Evaluation of natural killer cell killing ability in combination with an antibody against Ramos cells of NK92MI cells transduced with CAR (3rd generation).
  • 1D shows the expression levels of each CAR in NK92MI cells transduced with CD16V-Z CAR (1st generation) and CD16V-OX40LZ CAR (2nd generation) according to an embodiment of the invention.
  • Figure 1E shows the natural killer cell killing capacity of the NK92MI cells transduced with CD16V-Z CAR (1st generation) and CD16V-OX40LZ CAR (2nd generation) according to an embodiment of the present invention in combination with the antibody against Ramos cells. .
  • 1F shows the expression level of each CAR in NK92MI cells transduced with CD16V-Z CAR (1st generation) and CD16V-ZOX40L CAR (2nd generation) according to an embodiment of the invention.
  • Figure 1G shows the natural killer cell killing capacity of the NK92MI cells transduced with the CD16V-Z CAR (1st generation) and CD16V-ZOX40L CAR (2nd generation) according to the embodiment of the present invention in combination with the antibody against Ramos cells. .
  • FIG. 2A shows the expression of each CAR in NK92MI cells transduced with a CD16V-Z CAR (1st generation), CD16V-BBZ CAR (2nd generation), or CD16V-BBOX40LZ CAR (3rd generation) according to an embodiment of the invention. Indicates the amount.
  • 2B is a combination of antibodies against Ramos cells of NK92MI cells expressing CD16V-Z CAR (1st generation), CD16V-BBZ CAR (2nd generation), or CD16V-BBOX40LZ CAR (3rd generation) according to an embodiment of the present invention. Evaluation of natural killer cell killing ability.
  • Figure 3A shows the expression level of each CAR in NK92MI cells transduced with the third generation CARs CD16V-28OX40LZ CAR, CD16V-28OX40Z CAR, or CD16V-28BBZ CAR according to an embodiment of the present invention.
  • FIG. 3B shows a natural killer cell killing capacity of NK92MI cells expressing NK92MI cells expressing third generation CARs CD16V-28OX40LZ CAR, CD16V-28OX40Z CAR, or CD16V-28BBZ CAR according to an embodiment of the present invention. Indicates.
  • CD16V-28 (H) BBZ CAR, CD16V-28 (H) OX40Z CAR, or CD16V-28 (H) OX40LZ CAR which are third generation CARs including CD28 in the hinge according to an embodiment of the present invention.
  • the expression level of each CAR in the NK92MI cells was shown.
  • FIG. 4B shows CD16V-28 (H) BBZ CAR, CD16V-28 (H) OX40Z CAR, or CD16V-28 (H) OX40LZ CAR, which are third generation CARs comprising CD28 in a hinge according to an embodiment of the present invention. Evaluation of natural killer cell killing ability when NK92MI cells are used in combination with antibody against Ramos cells.
  • 5A is a NK92MI transduced with NKG2D-Z CAR (1st generation), NKG2D-28Z CAR (2nd generation), NKG2D-BBZ CAR (2nd generation), NKG2D-OX40Z CAR (2nd generation) according to an embodiment of the present invention.
  • the expression level of each CAR in the cell is shown.
  • 5B is a NK92MI transduced with NKG2D-Z CAR (1st generation), NKG2D-28Z CAR (2nd generation), NKG2D-BBZ CAR (2nd generation), NKG2D-OX40Z CAR (2nd generation) according to an embodiment of the present invention.
  • the natural killer cell killing ability of the human breast cancer cell line MCF-7 is shown.
  • FIG. 6A shows NK92MI cells transduced with NKG2D-Z CAR (1st generation), NKG2D-28BBZ CAR (3rd generation), and NKG2D-28OX40Z CAR (3rd generation) containing CD28 signaling domain according to an embodiment of the present invention. The expression amount of each CAR is shown.
  • FIG. 6B shows NK92MI cells transduced with NKG2D-Z CAR (1st generation), NKG2D-28BBZ CAR (3rd generation), and NKG2D-28OX40Z CAR (3rd generation) containing a CD28 signaling domain according to an embodiment of the present invention.
  • the natural killer cell killer ability evaluation of the human breast cancer cell line MCF-7 is shown.
  • FIG. 7A shows an NKG2D-Z CAR (1st generation), NKG2D-28Z CAR (2nd generation), NKG2D-28 (H) OX40LZ CAR (3rd generation), between an NKG2D extracellular domain and a CD28 hinge, according to an embodiment of the invention.
  • the expression level of each CAR in NK92MI cells transduced with NKG2D-AAA-28 (H) OX40LZ CAR (3rd generation) containing AAA sequence is shown.
  • FIG. 7B shows an NKG2D-Z CAR (1st generation), NKG2D-28Z CAR (2nd generation), NKG2D-28 (H) OX40LZ CAR (3rd generation), between the NKG2D extracellular domain and the CD28 hinge, according to an embodiment of the invention.
  • the NK92MI cells transduced with NKG2D-AAA-28 (H) OX40LZ CAR (3rd generation) containing the AAA sequence are shown to evaluate natural killer cell killing ability against human breast cancer cell line MCF-7.
  • the expression level of each CAR in the NK92MI cells was shown.
  • FIG. 8B shows expression levels of various NKG2D ligands in human lung cancer cell lines H1299 and H1944 according to an embodiment of the present invention.
  • NKG2D-AAA-28 H
  • OX40LZ CAR 3rd generation
  • an AAA sequence between the NKG2D-Z CAR (1st generation), the NKG2D extracellular domain and the CD28 hinge according to an embodiment of the present invention.
  • the present invention relates to a chimeric antigen receptor and natural killer cells expressing the same, and includes an intracellular signaling domain including all or a part of an OX40 ligand (CD252), thereby enhancing the anticancer activity of immune cells. It relates to a good chimeric antigen receptor (CAR) and immune cells expressing it.
  • CAR chimeric antigen receptor
  • Chimeric antigen receptors of the invention comprise an intracellular signaling domain comprising all or a portion of the OX40 ligand (CD252).
  • the chimeric antigen receptor comprises a transmembrane domain connected to the intracellular signaling domain; A spacer domain linked to the transmembrane domain; And an extracellular domain linked to the spacer domain.
  • the chimeric antigen receptor may further include a signal sequence connected to a terminal which is not connected to the spacer domain of the extracellular domain.
  • each of the above domains may be directly connected to each other or may be connected by a linker.
  • the signal sequence may be such that the extracellular domain can be located outside the cell membrane of immune cells (eg, natural killer cells) when the chimeric antigen receptor is expressed.
  • the signal sequence may include all or part of CD16.
  • the extracellular domain is a domain for binding specifically to the antibody or specifically recognize the antigen, for example Fc receptor (Fc receptor), single-chain variable fragment (single-chain variable fragment) , An antigen-binding fragment of an antibody such as ScFv), a natural cytotoxicity receptor, NKG2D, 2B4 or DNAM-1.
  • Fc receptor Fc receptor
  • single-chain variable fragment single-chain variable fragment
  • An antigen-binding fragment of an antibody such as ScFv
  • NKG2D, 2B4 or DNAM-1 a natural cytotoxicity receptor
  • the chimeric antigen receptor according to one embodiment of the present invention can be used with various antibodies depending on the cell type of the cancer to be treated by including the Fc receptor as an extracellular domain.
  • the Fc receptor may be any one selected from the group consisting of CD16, CD32, CD64, CD23 and CD89.
  • the Fc receptor may comprise all or part of the CD16 V158 variant (CD16V).
  • the chimeric antigen receptor of the present invention may include as an extracellular domain an antigen binding fragment of an antibody that directly recognizes the antigen as an extracellular domain without co-administration with the antibody.
  • the antigen-binding fragment may be a Fab fragment, F (ab ') fragment, F (ab') 2 fragment or Fv fragment.
  • the antibody may be any one of various kinds of antibodies capable of antigen-specific binding.
  • an antibody may be one light chain and one heavy chain combined, and two light chains and two heavy chains combined.
  • the first unit in which the first light chain and the first heavy chain are combined, and the second unit in which the second light chain and the second heavy chain are combined may be combined with each other.
  • the bond may be, but is not limited to, a disulfide bond.
  • the two units may be the same as or different from each other.
  • the first unit including the first light chain and the first heavy chain and the second unit including the second light chain and the second heavy chain may be the same as or different from each other.
  • an antibody prepared such that the first unit and the second unit can recognize two different antigens is commonly referred to in the art as a bispecific antibody.
  • the antibody may be a combination of three or more of the above units.
  • the antigen-binding fragment of the present invention may be derived from various types of antibodies as described above, but is not limited thereto.
  • the extracellular domain used in the present invention may be a natural cytotoxicity receptor.
  • the natural killer receptors include, but are not limited to, NKp46, NKp30, NKp44, NKp80 and NKp65 receptors.
  • the transmembrane domain is located through the cell membrane, and any one can be located through the cell membrane without interfering with the function of the extracellular domain and the intracellular signaling domain. Any use is available.
  • the transmembrane domain may include all or part of any one selected from the group consisting of CD8 ⁇ and CD28.
  • the extracellular domain and the transmembrane domain may be connected by a spacer domain.
  • the spacer domain can be a hinge domain.
  • the spacer domain may include all or part of any one selected from the group consisting of CD8 ⁇ and CD28.
  • the intracellular signaling domain is a part which is located inside the cell membrane of the natural killer cell, that is, the cytoplasm, when the antibody bound to the extracellular domain of the present invention binds to the target antigen, It may include a sequence capable of transmitting a signal for activating natural killer cells.
  • the chimeric antigen receptor may comprise one or more intracellular signaling domains.
  • the intracellular signaling domains may be connected in series with each other.
  • one end of the first intracellular signaling domain is connected to an end which is not connected to the spacer domain of the transmembrane domain, and the first intracellular signaling domain is connected.
  • One end of a second intracellular signaling domain is linked to a terminus that is not linked to the transmembrane domain of and a third to a terminus that is not linked to the first intracellular signaling domain of the second intracellular signaling domain.
  • One end of the intracellular signaling domain may be linked.
  • first, second and third intracellular signaling domains may be located in order in the cell membrane in the intracellular direction. Even if two, four or more intracellular signaling domains can be linked to one another in the same way. According to one embodiment of the invention, each of the above domains may be directly connected to each other or may be connected by a linker.
  • the chimeric antigen receptor may be one comprising two intracellular signaling domains.
  • it may include a first intracellular signaling domain connected to the transmembrane domain and a second intracellular signaling domain connected to a terminal not connected to the transmembrane domain of the first intracellular signaling domain.
  • the first intracellular signaling domain is OX40 (CD134), OX40 ligand (OX40L, CD252), 4-1BB (CD137), CD28, DAP10, CD3-zeta (CD3 ⁇ ) and DAP12 It may include all or part of any one selected from the group consisting of, wherein the second intracellular signaling domain comprises all or part of any one selected from the group consisting of OX40 ligand, CD3-zeta and DAP12 It may be. In this case, at least one of the first intracellular signaling domain and the second intracellular signaling domain comprises all or part of the OX40 ligand.
  • the chimeric antigen receptor may comprise a first intracellular signaling domain comprising all or part of an OX40 ligand and a second intracellular signaling comprising all or part of any one selected from the group consisting of CD3-zeta and DAP12. It may be to include a domain. Also, for example, the chimeric antigen receptor may comprise a first intracellular signaling domain comprising all or part of any one selected from the group consisting of CD3-zeta and DAP12 and a second intracellular signal comprising all or part of an OX40 ligand. It may be to include a delivery domain.
  • the chimeric antigen receptor may comprise three intracellular signaling domains. For example, a first intracellular signaling domain linked to the transmembrane domain; A second intracellular signaling domain linked to a terminus not linked to said transmembrane domain of said first intracellular signaling domain; And a third intracellular signaling domain connected to a terminal which is not connected to the first intracellular signaling domain of the second intracellular signaling domain.
  • the first intracellular signaling domain may include all or a part of any one selected from the group consisting of 4-1BB, OX40, OX40 ligand, CD28 and DAP10
  • the agent 2 the intracellular signaling domain may comprise all or part of any one selected from the group consisting of OX40 ligand, OX40 and 4-1BB
  • the third intracellular signaling domain may comprise all or part of any one selected from the group consisting of OX40 ligand, CD3-zeta and DAP12.
  • at least one of the first intracellular signaling domain, the second intracellular signaling domain, and the third intracellular signaling domain comprises all or part of the OX40 ligand.
  • the first intracellular signaling domain comprising all or part of any one selected from the group consisting of CD28 and 4-1BB; A second intracellular signaling domain comprising all or a portion of any one selected from the group consisting of an OX40 ligand, OX40 and 4-1BB; And a third intracellular signaling domain comprising all or a portion of CD3-zeta, wherein the first, second and third intracellular signaling domains are located in sequence in the intracellular direction at the cell membrane.
  • each of the above domains may be directly connected to each other or may be connected by a linker.
  • the chimeric antigen receptor comprises a transmembrane domain (transmembrane domain) connected to the first intracellular signaling domain; A spacer domain linked to the transmembrane domain; And an extracellular domain linked to the spacer domain.
  • the chimeric antigen receptor may further include a signal sequence connected to the extracellular domain.
  • each of the above domains may be directly connected to each other or may be connected by a linker.
  • the extracellular domain is a domain for binding specifically to the antibody or specifically recognize the antigen, for example Fc receptor (Fc receptor), single-chain variable fragment (single-chain variable fragment) , An antigen-binding fragment of an antibody such as ScFv), a natural cytotoxicity receptor, NKG2D, 2B4 or DNAM-1.
  • Fc receptor Fc receptor
  • single-chain variable fragment single-chain variable fragment
  • An antigen-binding fragment of an antibody such as ScFv
  • NKG2D, 2B4 or DNAM-1 a natural cytotoxicity receptor
  • the chimeric antigen receptor according to one embodiment of the present invention can be used with various antibodies depending on the cell type of the cancer to be treated by including the Fc receptor as an extracellular domain.
  • the Fc receptor may be any one selected from the group consisting of CD16, CD32, CD64, CD23, CD89 and variants thereof.
  • the Fc receptor may comprise a CD16 or a variant thereof, and most specifically, may include all or a part of the CD16 V158 variant (CD16V).
  • the chimeric antigen receptor of the present invention may include as an extracellular domain an antigen binding fragment of an antibody that directly recognizes the antigen as an extracellular domain without co-administration with the antibody.
  • the antigen-binding fragment may be a Fab fragment, F (ab ') fragment, F (ab') 2 fragment or Fv fragment.
  • the antibody may be any one of various kinds of antibodies capable of antigen-specific binding.
  • an antibody may be one light chain and one heavy chain combined, and two light chains and two heavy chains combined.
  • the first unit in which the first light chain and the first heavy chain are combined, and the second unit in which the second light chain and the second heavy chain are combined may be combined with each other.
  • the bond may be, but is not limited to, a disulfide bond.
  • the two units may be the same as or different from each other.
  • the first unit including the first light chain and the first heavy chain and the second unit including the second light chain and the second heavy chain may be the same as or different from each other.
  • an antibody prepared such that the first unit and the second unit can recognize two different antigens is commonly referred to in the art as a bispecific antibody.
  • the antibody may be a combination of three or more of the above units.
  • the antigen-binding fragment of the present invention may be derived from various types of antibodies as described above, but is not limited thereto.
  • the extracellular domain used in the present invention may be a natural cytotoxicity receptor.
  • the natural killer receptors include, but are not limited to, NKp46, NKp30, NKp44, NKp80 and NKp65 receptors.
  • the signal sequence may include all or a part of CD16.
  • the extracellular domain may include all or a portion of the CD16 V158 variant (CD16V).
  • the spacer domain may include all or part of any one selected from the group consisting of CD8 ⁇ (CD8-alpha) and CD28.
  • the transmembrane domain may include all or part of any one selected from the group consisting of CD8 ⁇ and CD28.
  • the chimeric antigen receptor is SEQ ID NO: 33, 41, 43, 45, 47, 49, 51, 53, 55, 69, 71, 77, 81, 83, 85, 87, 89, 91, and 93 It may include one or more amino acid sequences selected from the group consisting of or variants thereof having 80% or more sequence identity.
  • the present invention further provides immune cells (eg, natural killer cells (NK cells)) expressing the chimeric antigen receptor according to the invention described above.
  • immune cells eg, natural killer cells (NK cells)
  • NK cells natural killer cells
  • the immune cells of the present invention may be toxic to tumor cells. Since the chimeric antigen receptor according to the present invention is determined which tumor cell is specifically toxic depending on which antibody the extracellular domain binds to, the immune cells expressing the chimeric antigen receptor according to the present invention are toxic. Tumor cells that can be expressed are not particularly limited. According to one embodiment, when the immune cells of the present invention (eg, natural killer cells) are used with rituximab, they may be toxic to malignant lymphoma cells.
  • the malignant lymphoma cells may be expressing CD20. Also for example, the malignant lymphoma may be B-cell lymphoma.
  • the present invention further provides that the number of immune cells (eg, natural killer cells) expressing chimeric antigen receptors according to the invention described above is comprised between two and 7.5 times the number of tumor cells (eg, malignant lymphoma cells) in the subject of treatment, It provides a pharmaceutical composition for the prevention or treatment of a tumor or tumor metastasis.
  • immune cells eg, natural killer cells
  • tumor cells eg, malignant lymphoma cells
  • the pharmaceutical composition of the present invention comprises from 0.75 times the number of the immune cells (eg, natural killer cells) in one dose to the number of the tumor cells (eg, malignant lymphoma cells) in the treatment target. It may be included 10 times.
  • the number of immune cells (eg, natural killer cells) in a single dose may be comprised between 2 and 7.5 times the number of the tumor cells (eg, malignant lymphoma cells) in the treatment subject.
  • the present invention also provides a nucleic acid sequence encoding the chimeric antigen receptor according to the invention described above.
  • the nucleic acid sequence is SEQ ID NO: 32, 40, 42, 44, 46, 48, 50, 52, 54, 68, 70, 76, 80, 82, 84, 86, 88, 90 and At least one nucleotide sequence selected from the group consisting of 92 or variants thereof having at least 80% sequence identity.
  • the present invention also provides a vector comprising the nucleic acid sequence according to the present invention described above.
  • the present invention also provides a method for treating a tumor comprising administering the above-described immune cells to a subject.
  • the present invention also provides a method for preventing tumor metastasis, comprising administering the above-described immune cells to a subject.
  • the subject may be a mammal with a tumor, specifically, a human, but is not limited thereto.
  • Administration can be in an amount of 2 to 7.5 times the number of immune cells (eg, natural killer cells) expressing the chimeric antigen receptor according to the invention, of the number of tumor cells (eg, malignant lymphoma cells) in the treatment subject.
  • immune cells eg, natural killer cells
  • tumor cells eg, malignant lymphoma cells
  • the method of administration is not particularly limited and may be administered via conventional oral or parenteral routes.
  • the tumor is not particularly limited, and may be, for example, malignant lymphoma, leukemia, breast cancer, lung cancer, or the like, and more specifically, B-cell lymphoma.
  • Human B-family cell line Ramos, human erythroleukemic cell line K562, human breast cancer cell line MCF-7, human lung cancer cell line H1299 and H1944 cell line and NK-92MI are supplied from American Type Culture Collection, Manassas, VA, USA Was used. K562 was maintained on RPMI-1640 (Gibco, Grand Island, NY, USA) containing 10% FBS. Ramos was maintained in RPMI-1640 (ATCC) (Manassas, VA) containing 10% FBS (fetal bovine serum; Gibco, Grand Island, NY, USA).
  • MCF-7 was maintained in EMEM (ATCC) + 10% FBS (Gibco) medium and H1299, H1944 cell lines were maintained in RPMI-1640 (ATCC) + 10% FBS (Gibco).
  • NK-92MI and transformed NK-92MI cells were maintained in CellGro® serum free medium containing 1% human plasma.
  • Human embryonic kidney fibroblast 293T cell line was supplied from ATCC and used in DMEM (Gibco, Grand Island, NY, USA) containing 10% FBS (Gibco, Grand Island, NY, USA). Maintained.
  • FCRG3A V158 variant CD16V
  • NKG2D extracellular domain Signal sequence of CD8 ⁇ , hinge and transmembrane domain of CD8 ⁇ ; Hinge and transmembrane domains of CD28;
  • the intracellular signaling domains of 4-1BB, OX40, OX40 ligand (OX40L) and CD3 ⁇ were artificially synthesized, respectively. They were assembled in various combinations using SOE-PCR (splicing by overlapping extension by PCR). PCR results were confirmed by direct sequencing.
  • PCR results were digested with Nhe1 and EcoRI and then inserted into the Nhe1 and EcoRI sites of the MSCV-EF1 ⁇ -GFP vector or EF1a-MCS vector, a third generation self-inactivating lentiviral expression vector. ligation).
  • CAR chimeric antigen receptor
  • CD16V-Z CAR (1st generation) comprises the signal sequence domain of CD16 (34-84 nucleotides, GenBank Accession No. X52645); Extracellular domain of CD16V (FCRG3A V158) (85-651 nucleotides, G mutation of 559 nucleotides in GenBank Accession No. X52645); Hinge and transmembrane domains derived from human CD8 ⁇ (1292-1507 nucleotides, GenBank NM 001768.6); And a stop codon TGA linked to the intracellular signaling domain (299-634 nucleotides, GenBank NM000734.3) derived from CD3 ⁇ .
  • CD16V-BBZ CAR (2nd generation) comprises the signal sequence domain of CD16 (34-84 nucleotides, GenBank Accession No. X52645); Extracellular domain of CD16V (FCRG3A V158) (85-651 nucleotides, G mutation of 559 nucleotides in GenBank Accession No.
  • X52645 Hinge and transmembrane domains derived from human CD8 ⁇ (1292-1507 nucleotides, GenBank NM 001768.6); Intracellular signaling domains derived from CD137 (901-1026 nucleotides, GenBank NM 001561.5); And a stop codon TGA linked to the intracellular signaling domain (299-634 nucleotides, GenBank NM000734.3) derived from CD3 ⁇ .
  • CD16V-OX40Z CAR (2nd generation) comprises the signal sequence domain of CD16 (34-84 nucleotides, GenBank Accession No. X52645); Extracellular domain of CD16V (FCRG3A V158) (85-651 nucleotides, G mutation of 559 nucleotides in GenBank Accession No.
  • X52645 Hinge and transmembrane domains derived from human CD8 ⁇ (1292-1507 nucleotides, GenBank NM 001768.6); Intracellular signaling domains derived from CD134 (733-840 nucleotides, GenBank AB590584.1); And a stop codon TGA linked to the intracellular signaling domain (299-634 nucleotides, GenBank NM000734.3) derived from CD3 ⁇ .
  • CD16V-OX40LZ CAR (2nd generation) comprises the signal sequence domain of CD16 (34-84 nucleotides, GenBank Accession No. X52645); Extracellular domain of CD16V (FCRG3A V158) (85-651 nucleotides, G mutation of 559 nucleotides in GenBank Accession No.
  • X52645 Hinge and transmembrane domains derived from human CD8 ⁇ (1292-1507 nucleotides, GenBank NM 001768.6); Intracellular signaling domains derived from CD252 (141-206 nucleotides, GenBank NM 003326.4); And a stop codon TGA linked to the intracellular signaling domain (299-634 nucleotides, GenBank NM000734.3) derived from CD3 ⁇ .
  • CD16V-ZOX40L CAR (2nd generation) comprises the signal sequence domain of CD16 (34-84 nucleotides, GenBank Accession No. X52645); Extracellular domain of CD16V (FCRG3A V158) (85-651 nucleotides, G mutation of 559 nucleotides in GenBank Accession No.
  • CD16V-28Z CAR (2nd generation) is a signal sequence domain (34-84 nucleotides, GenBank Accession) of CD16. No.
  • X52645 Extracellular domain of CD16V (FCRG3A V158) (85-651 nucleotides, G mutation of 559 nucleotides in GenBank Accession No. X52645); Hinge from human CD8 ⁇ (1292-1435 nucleotides, GenBank NM 001768.6); Transmembrane and intracellular signaling domains derived from CD28 (679-882 nucleotides, GenBank NM 006139.3); And a stop codon TGA linked to the intracellular signaling domain (299-634 nucleotides, GenBank NM000734.3) derived from CD3 ⁇ .
  • CD16V-28 (H) Z CAR (2nd generation) comprises the signal sequence domain of CD16 (34-84 nucleotides, GenBank Accession No. X52645); Extracellular domain of CD16V (FCRG3A V158) (85-651 nucleotides, G mutation of 559 nucleotides in GenBank Accession No. X52645); Hinge, transmembrane and intracellular signaling domains derived from CD28 (562-882 nucleotides, GenBank NM 006139.3); And a stop codon TGA linked to the intracellular signaling domain (299-634 nucleotides, GenBank NM000734.3) derived from CD3 ⁇ .
  • CD16V-BBOX40Z CAR (3rd generation) comprises the signal sequence domain of CD16 (34-84 nucleotides, GenBank Accession No. X52645); Extracellular domain of CD16V (FCRG3A V158) (85-651 nucleotides, G mutation of 559 nucleotides in GenBank Accession No.
  • X52645 Hinge and transmembrane domains derived from human CD8 ⁇ (1292-1507 nucleotides, GenBank NM 001768.6); Intracellular signaling domains derived from CD137 (901-1026 nucleotides, GenBank NM 001561.5); Intracellular signaling domains derived from CD134 (733-840 nucleotides, GenBank AB590584.1); And a stop codon TGA linked to the intracellular signaling domain (299-634 nucleotides, GenBank NM000734.3) derived from CD3 ⁇ .
  • the CD16V-OX40BBZ CAR (3rd generation) comprises the signal sequence domain of CD16 (34-84 nucleotides, GenBank Accession No. X52645); Extracellular domain of CD16V (FCRG3A V158) (85-651 nucleotides, G mutation of 559 nucleotides in GenBank Accession No.
  • X52645 Hinge and transmembrane domains derived from human CD8 ⁇ (1292-1507 nucleotides, GenBank NM 001768.6); Intracellular signaling domains derived from CD134 (733-840 nucleotides, GenBank AB590584.1); Intracellular signaling domains derived from CD137 (901-1026 nucleotides, GenBank NM 001561.5); And a stop codon TGA linked to the intracellular signaling domain (299-634 nucleotides, GenBank NM000734.3) derived from CD3 ⁇ .
  • CD16V-28BBZ CAR (3rd generation) comprises the signal sequence domain of CD16 (34-84 nucleotides, GenBank Accession No. X52645); Extracellular domain of CD16V (FCRG3A V158) (85-651 nucleotides, G mutation of 559 nucleotides in GenBank Accession No.
  • CD8 ⁇ (1292-1435 nucleotides, GenBank NM 001768.6)
  • Transmembrane and intracellular signaling domains derived from CD28 (679-882 nucleotides, GenBank NM 006139.3)
  • Intracellular signaling domains derived from CD137 (901-1026 nucleotides, GenBank NM 001561.5)
  • a stop codon TGA linked to the intracellular signaling domain (299-634 nucleotides, GenBank NM000734.3) derived from CD3 ⁇ .
  • the CD16V-28OX40Z CAR (3rd generation) comprises the signal sequence domain of CD16 (34-84 nucleotides, GenBank Accession No. X52645); Extracellular domain of CD16V (FCRG3A V158) (85-651 nucleotides, G mutation of 559 nucleotides in GenBank Accession No.
  • CD8 ⁇ (1292-1435 nucleotides, GenBank NM 001768.6); Transmembrane and intracellular signaling domains derived from CD28 (679-882 nucleotides, GenBank NM 006139.3); Intracellular signaling domains derived from CD134 (733-840 nucleotides, GenBank AB590584.1); And a stop codon TGA linked to the intracellular signaling domain (299-634 nucleotides, GenBank NM000734.3) derived from CD3 ⁇ .
  • CD16V-28OX40LZ CAR (3rd generation) comprises the signal sequence domain of CD16 (34-84 nucleotides, GenBank Accession No. X52645); Extracellular domain of CD16V (FCRG3A V158) (85-651 nucleotides, G mutation of 559 nucleotides in GenBank Accession No.
  • CD8 ⁇ (1292-1435 nucleotides, GenBank NM 001768.6); Transmembrane and intracellular signaling domains derived from CD28 (679-882 nucleotides, GenBank NM 006139.3); Intracellular signaling domains derived from CD252 (141-206 nucleotides, GenBank NM 003326.4); And a stop codon TGA linked to the intracellular signaling domain (299-634 nucleotides, GenBank NM000734.3) derived from CD3 ⁇ .
  • CD16V-28 (H) BBZ CAR (3rd generation) comprises the signal sequence domain of CD16 (34-84 nucleotides, GenBank Accession No. X52645); Extracellular domain of CD16V (FCRG3A V158) (85-651 nucleotides, G mutation of 559 nucleotides in GenBank Accession No.
  • CD16V-28 (H) OX40Z CAR (3rd generation) comprises the signal sequence domain of CD16 (34-84 nucleotides, GenBank Accession No. X52645); Extracellular domain of CD16V (FCRG3A V158) (85-651 nucleotides, G mutation of 559 nucleotides in GenBank Accession No.
  • CD16V-28 (H) OX40LZ CAR (3rd generation) comprises the signal sequence domain of CD16 (34-84 nucleotides, GenBank Accession No. X52645); Extracellular domain of CD16V (FCRG3A V158) (85-651 nucleotides, G mutation of 559 nucleotides in GenBank Accession No.
  • CD16V-BBOX40LZ (3rd generation) is the signal sequence domain of CD16 (34-84 nucleotides, GenBank Accession No. X52645); Extracellular domain of CD16V (FCRG3A V158) (85-651 nucleotides, G mutation of 559 nucleotides in GenBank Accession No.
  • X52645 Hinge and transmembrane domains derived from human CD8 ⁇ (1292-1507 nucleotides, GenBank NM 001768.6); Intracellular signaling domains derived from CD137 (901-1026 nucleotides, GenBank NM 001561.5); Intracellular signaling domains derived from CD252 (141-206 nucleotides, GenBank NM 003326.4); And a stop codon TGA linked to the intracellular signaling domain (299-634 nucleotides, GenBank NM000734.3) derived from CD3 ⁇ .
  • CAR chimeric antigen receptor
  • CAR chimeric antigen receptor
  • NKG2D-Z CAR (1st generation) comprises the signal sequence domain of CD8 ⁇ (890-952 nucleotides, GenBank NM 001768.6); Extracellular domain of NKG2D (788-1192 nucleotides, GenBank ID: AF461811.1); Hinge and transmembrane domains derived from human CD8 ⁇ (1292-1507 nucleotides, GenBank NM 001768.6); And a stop codon TGA linked to the intracellular signaling domain (299-634 nucleotides, GenBank NM000734.3) derived from CD3 ⁇ .
  • NKG2D-BBZ CAR (2nd generation) comprises the signal sequence domain of CD8 ⁇ (890-952 nucleotides, GenBank NM 001768.6); Extracellular domain of NKG2D (788-1192 nucleotides, GenBank ID: AF461811.1); Hinge and transmembrane domains derived from human CD8 ⁇ (1292-1507 nucleotides, GenBank NM 001768.6); Intracellular signaling domains derived from CD137 (901-1026 nucleotides, GenBank NM 001561.5); And a stop codon TGA linked to the intracellular signaling domain (299-634 nucleotides, GenBank NM000734.3) derived from CD3 ⁇ .
  • NKG2D-OX40Z CAR (2nd generation) contains the signal sequence domain of CD8 ⁇ (890-952 nucleotides, GenBank NM 001768.6); Extracellular domain of NKG2D (788-1192 nucleotides, GenBank ID: AF461811.1); Hinge and transmembrane domains derived from human CD8 ⁇ (1292-1507 nucleotides, GenBank NM 001768.6); Intracellular signaling domains derived from CD134 (733-840 nucleotides, GenBank AB590584.1); And a stop codon TGA linked to the intracellular signaling domain (299-634 nucleotides, GenBank NM000734.3) derived from CD3 ⁇ .
  • NKG2D-ZOX40L CAR contains the signal sequence domain of CD8 ⁇ (890-952 nucleotides, GenBank NM 001768.6); Extracellular domain of NKG2D (788-1192 nucleotides, GenBank ID: AF461811.1); Hinge and transmembrane domains derived from human CD8 ⁇ (1292-1507 nucleotides, GenBank NM 001768.6); CD3 ⁇ -derived intracellular signaling domain (299-634 nucleotides, GenBank NM000734.3) and CD252-derived intracellular signaling domain (141-206 nucleotides, GenBank NM 003326.4) are linked to the CD3 ⁇ stop codon TGA.
  • NKG2D-28Z CAR (2nd generation) contains the signal sequence domain of CD8 ⁇ (890-952 nucleotides, GenBank NM 001768.6); Extracellular domain of NKG2D (788-1192 nucleotides, GenBank ID: AF461811.1); Hinge from human CD8 ⁇ (1292-1435 nucleotides, GenBank NM 001768.6); Transmembrane and intracellular signaling domains derived from CD28 (679-882 nucleotides, GenBank NM 006139.3); And a stop codon TGA linked to the intracellular signaling domain (299-634 nucleotides, GenBank NM000734.3) derived from CD3 ⁇ .
  • NKG2D-28 (H) Z CAR (2nd generation) comprises the signal sequence domain of CD8 ⁇ (890-952 nucleotides, GenBank NM 001768.6); Extracellular domain of NKG2D (788-1192 nucleotides, GenBank ID: AF461811.1); Hinge, transmembrane and intracellular signaling domains derived from CD28 (562-882 nucleotides, GenBank NM 006139.3); And a stop codon TGA linked to the intracellular signaling domain (299-634 nucleotides, GenBank NM000734.3) derived from CD3 ⁇ .
  • NKG2D-BBOX40Z CAR (3rd generation) comprises the signal sequence domain of CD8 ⁇ (890-952 nucleotides, GenBank NM 001768.6); Extracellular domain of NKG2D (788-1192 nucleotides, GenBank ID: AF461811.1); Hinge and transmembrane domains derived from human CD8 ⁇ (1292-1507 nucleotides, GenBank NM 001768.6); Intracellular signaling domains derived from CD137 (901-1026 nucleotides, GenBank NM 001561.5); Intracellular signaling domains derived from CD134 (733-840 nucleotides, GenBank AB590584.1); And a stop codon TGA linked to the intracellular signaling domain (299-634 nucleotides, GenBank NM000734.3) derived from CD3 ⁇ .
  • NKG2D-BBOX40LZ CAR (3rd generation) is the signal sequence domain of CD8 ⁇ (890-952 nucleotides, GenBank NM 001768.6); Extracellular domain of NKG2D (788-1192 nucleotides, GenBank ID: AF461811.1); Hinge and transmembrane domains derived from human CD8 ⁇ (1292-1507 nucleotides, GenBank NM 001768.6); Intracellular signaling domains derived from CD137 (901-1026 nucleotides, GenBank NM 001561.5); Intracellular signaling domains derived from CD252 (141-206 nucleotides, GenBank NM 003326.4); And a stop codon TGA linked to the intracellular signaling domain (299-634 nucleotides, GenBank NM000734.3) derived from CD3 ⁇ .
  • NKG2D-OX40BBZ CAR (3rd generation) is a signal sequence domain of CD8 ⁇ (890-952 nucleotides, GenBank NM 001768.6); Extracellular domain of NKG2D (788-1192 nucleotides, GenBank ID: AF461811.1); Hinge and transmembrane domains derived from human CD8 ⁇ (1292-1507 nucleotides, GenBank NM 001768.6); Intracellular signaling domains derived from CD134 (733-840 nucleotides, GenBank AB590584.1); Intracellular signaling domains derived from CD137 (901-1026 nucleotides, GenBank NM 001561.5); And a stop codon TGA linked to the intracellular signaling domain (299-634 nucleotides, GenBank NM000734.3) derived from CD3 ⁇ .
  • NKG2D-28BBZ CAR (3rd generation) is a signal sequence domain of CD8 ⁇ (890-952 nucleotides, GenBank NM 001768.6); Extracellular domain of NKG2D (788-1192 nucleotides, GenBank ID: AF461811.1); Hinge domain from CD8 ⁇ (1292-1435 nucleotides, GenBank NM 001768.6); Transmembrane and intracellular signaling domains derived from CD28 (679-882 nucleotides, GenBank NM 006139.3); Intracellular signaling domains derived from CD137 (901-1026 nucleotides, GenBank NM 001561.5); And a stop codon TGA linked to the intracellular signaling domain (299-634 nucleotides, GenBank NM000734.3) derived from CD3 ⁇ .
  • the NKG2D-28OX40Z CAR (3rd generation) contains the signal sequence domain of CD8 ⁇ (890-952 nucleotides, GenBank NM 001768.6); Extracellular domain of NKG2D (788-1192 nucleotides, GenBank ID: AF461811.1); Hinge domain from CD8 ⁇ (1292-1435 nucleotides, GenBank NM 001768.6); Transmembrane and intracellular signaling domains derived from CD28 (679-882 nucleotides, GenBank NM 006139.3); Intracellular signaling domains derived from CD134 (733-840 nucleotides, GenBank AB590584.1); And a stop codon TGA linked to the intracellular signaling domain (299-634 nucleotides, GenBank NM000734.3) derived from CD3 ⁇ .
  • NKG2D-28OX40LZ CAR contains the signal sequence domain of CD8 ⁇ (890-952 nucleotides, GenBank NM 001768.6); Extracellular domain of NKG2D (788-1192 nucleotides, GenBank ID: AF461811.1); Hinge domain from CD8 ⁇ (1292-1435 nucleotides, GenBank NM 001768.6); Transmembrane and intracellular signaling domains derived from CD28 (679-882 nucleotides, GenBank NM 006139.3); Intracellular signaling domains derived from CD252 (141-206 nucleotides, GenBank NM 003326.4); And a stop codon TGA linked to the intracellular signaling domain (299-634 nucleotides, GenBank NM000734.3) derived from CD3 ⁇ .
  • NKG2D-28 (H) BBZ CAR (3rd generation) comprises the signal sequence domain of CD8 ⁇ (890-952 nucleotides, GenBank NM 001768.6); Extracellular domain of NKG2D (788-1192 nucleotides, GenBank ID: AF461811.1); Hinge, transmembrane and intracellular signaling domains derived from CD28 (562-882 nucleotides, GenBank NM 006139.3); Intracellular signaling domains derived from CD137 (901-1026 nucleotides, GenBank NM 001561.5); And a stop codon TGA linked to the intracellular signaling domain (299-634 nucleotides, GenBank NM000734.3) derived from CD3 ⁇ .
  • NKG2D-28 (H) OX40Z CAR (3rd generation) contains the signal sequence domain of CD8 ⁇ (890-952 nucleotides, GenBank NM 001768.6); Extracellular domain of NKG2D (788-1192 nucleotides, GenBank ID: AF461811.1); Hinge, transmembrane and intracellular signaling domains derived from CD28 (562-882 nucleotides, GenBank NM 006139.3); Intracellular signaling domains derived from CD134 (733-840 nucleotides, GenBank AB590584.1); And a stop codon TGA linked to the intracellular signaling domain (299-634 nucleotides, GenBank NM000734.3) derived from CD3 ⁇ .
  • NKG2D-28 (H) OX40LZ CAR (3rd generation) contains the signal sequence domain of CD8 ⁇ (890-952 nucleotides, GenBank NM 001768.6); Extracellular domain of NKG2D (788-1192 nucleotides, GenBank ID: AF461811.1); Hinge, transmembrane and intracellular signaling domains derived from CD28 (562-882 nucleotides, GenBank NM 006139.3); Intracellular signaling domains derived from CD252 (141-206 nucleotides, GenBank NM 003326.4); And a stop codon TGA linked to the intracellular signaling domain (299-634 nucleotides, GenBank NM000734.3) derived from CD3 ⁇ .
  • NKG2D-AAA-28 (H) OX40LZ CAR (3rd generation) contains the signal sequence domain of CD8 ⁇ (890-952 nucleotides, GenBank NM 001768.6); Extracellular domain of NKG2D (788-1192 nucleotides, GenBank ID: AF461811.1); Triple alanine (AAA); Hinge, transmembrane and intracellular signaling domains derived from CD28 (562-882 nucleotides, GenBank NM 006139.3); Intracellular signaling domains derived from CD252 (141-206 nucleotides, GenBank NM 003326.4); And a stop codon TGA linked to the intracellular signaling domain (299-634 nucleotides, GenBank NM000734.3) derived from CD3 ⁇ .
  • CAR chimeric antigen receptor
  • VSVG-pseudotyped lentiviral 293T cells cultured in DMEM medium were subjected to various types of PCDH1-MSCV-CD16-construct-EF1-copGFP vectors, EF1a-NKG2D-construct vectors or PCDH1-MSCV-EF1 -copGFP control vector, EF1a-GFP control vector (for production of mole infection virus using a blank vector); And co-transfected with the HIV-based pPACKH1 lentiviral package kit (System Biosciences), for which lipofectamine 2000 reagent (Invitrogen, Carlsbad, Calif.) Was used.
  • HIV-based pPACKH1 lentiviral package kit System Biosciences
  • CD16V constructs are as follows; CD16V-Z CAR, CD16V-BBZ CAR, CD16V-OX40Z CAR, CD16V-OX40LZ CAR, CD16V-ZOX40L, CD16V-28Z CAR, CD16V-28 (H) Z CAR, CD16V-BBOX40Z CAR, CD16V-BBOX40LZ CAR, CD16V- OX40BBZ CAR, CD16V-28BBZ CAR, CD16V-28OX40Z CAR, CD16V-28OX40LZ CAR, CD16V-28 (H) BBZ CAR, CD16V-28 (H) OX40Z CAR, CD16V-28 (H) OX40LZ CAR.
  • NKG2D constructs are as follows; NKG2D-Z CAR, NKG2D-BBZ CAR, NKG2D-OX40Z CAR, NKG2D-ZOX40L CAR, NKG2D-28Z CAR, NKG2D-28 (H) Z CAR, NKG2D-BBOX40Z CAR, NKG2D-BBOX40LZ CAR, NKG2D-OX40BBZ CAR, NKG2D -28BBZ CAR, NKG2D-28OX40Z CAR, NKG2D-28OX40LZ CAR, NKG2D-28 (H) BBZ CAR, NKG2D-28 (H) OX40Z CAR, NKG2D-28 (H) OX40LZ CAR, NKG2D-AAA-28 (H) OX40LZ .
  • Lentiviruses include various types of CD16V construct expression vectors or control plasmids in HEK293T cells that are about 80% dense in the flask; And pPACKH1 lentivirus packaging plasmids. After 6 hours the medium was replaced with DMEM medium containing 10% FBS. Conditioned medium containing lentiviral was collected 48 hours after transfection and filtered using a 0.45 ⁇ m filter unit (Milliopore, Billerica, MA, USA) to remove cell debris. The virus supernatant was concentrated about 50-fold by centrifugation at 3000 rpm and 4 ° C. for 20 minutes using Amicon Filter (Millipore). The concentrated virus was stored at -80 ° C.
  • NK92MI cells in the logarithmic phase were adjusted to a concentration of 1 ⁇ 10 6 cells / ml using Cellgro (Cellgenix) containing 1% human plasma, after which the lentiviral supernatant was reduced to 50-100 MOI.
  • Cellgro Cellgenix
  • CD16V CAR-transduced NK92MI cells NKG2D CAR-transduced NK92MI cells, control vector-transduced NK92MI (NK92MI-Mock), or NK-92MI parent cells were washed twice with FACS buffer and cells were washed 7 Staining was performed using -AAD (Beckman coulter), anti-CD3, anti-CD56 and anti-CD16 (BD Biosciences) mAbs. Expression ratio and mean fluorescence intensity (MFI) of the stained cells were measured using BD LSRFortessa.
  • Transformation efficiency using NKG2D constructs was measured by flow cytometry of NKG2D expressing cells among CD3-CD56 + cells. NK92MI cells were first gated for singlets and then gated for 7AAD- and CD3-CD56 +. Transformation efficiency using CD16 constructs was measured by flow cytometry of GFP and CD16 expressing cells among CD3-CD56 + cells.
  • Target cells were labeled for 1 hour at 37 ° C. using 30 ⁇ M calceinacetoxymethyl ester (Calcein-AM; Molecular probes). After washing, labeled target cells were dispensed into 1 x 104 cells per well in 96-well plates. NK92MI cells were harvested, washed and added with or without varying effector-to-target (E / T) ratios and varying concentrations of rituximab. As a control, anti-human antibody (Sigma aldrich) independent of rituximab was used.
  • Example 2 comprising OX40 ligand (CD252) chimera CD20-positive of NK92MI cells expressing antigen receptor (CAR) Lymphoma Cytotoxicity Assessment on Cells
  • the V158 variant of FCRG3A (CD16) is a high affinity immunoglobulin Fc receptor and is thought to have a good effect on antibody treatment.
  • CD16V containing chimeric antigen receptors (CD16V-Z CAR (1st generation), CD16V-28Z CAR (2nd generation), CD16V-BBZ CAR (2nd generation), CD16V-OX40Z CAR (2nd generation), or CD16V-28OX40LZ) CAR (3rd generation)) was expressed in NK92MI cells using a lentiviral vector comprising an MSCV promoter. Whether individual CARs are expressed on the surface of NK92MI cells was confirmed by detecting human CD16 using monoclonal mouse anti-human antibodies. By repeated experiments using flow cytometry, it was confirmed that CARs were transduced in NK92MI cells with an efficiency of 90% or more (FIG. 1A). The amount of the lentiviral vector used was 50 or more of multiples of infection (MOI). .
  • MOI multiples of infection
  • CD252 OX40 ligand
  • CD20-positive lymphoma cells Ros cells
  • NK92MI cells transduced with vectors only and NK92MI cells expressing a CD16V containing receptor Cytotoxicity against was assessed using the Calcein-AM release assay.
  • the intrinsic cell killing capacity of the transduced NK92MI cells was assessed using K562, a human erythroleukemic cell line, as a standard control.
  • MOI multiple of infection
  • individual CARs according to the invention CD16V-Z CAR (1st generation), CD16V-28Z CAR (2nd generation), CD16V-BBZ) CAR (2nd generation), CD16V-OX40Z CAR (2nd generation), or CD16V-28OX40LZ CAR (3rd generation) were expressed at high levels in at least 90% of NK92MI cells (FIG. 1A).
  • Cytotoxicity against K562 of NK92MI cells expressing CARs according to the present invention was shown to be similar to the cell killing ability against K562 of the control (Mock) transduced with the vector only. This means that the cell killing ability of the NK92MI cells to the target was not increased by side effects due to the genetic modification itself (FIG. 1B).
  • 5: 1, 2.5: 1, 1: 1, and 0.5: 1 represent a ratio of the number of effector cells, NK92MI cells, and the target cells, K562 cells.
  • NK92MI cells expressing CD16V containing receptors
  • B-cell lymphoma cell line transduced NK92MI cells and cancer cells (B-cell lymphoma cell line).
  • Phosphorus Ramos was assessed using Calcein-AM release assay after co-culture ( Figure 1C).
  • Fig. 1C 5: 1, 2.5: 1, 1: 1, and 0.5: 1 represent ratios of the number of effector cells, NK92MI cells, and the target cells, Ramos cells. Since NK92MI cells themselves do not express CD16, it is known that apoptosis by NK92MI does not increase even if rituximab is present.
  • CD16V-Z CAR 1st generation
  • CD16V-OX40Z CAR (NK2) CD16V-OX40Z CAR
  • NK2 CD16V-OX40Z CAR
  • NK-92MI cells expressing CD16V-28OX40LZ CAR (3rd generation) have strong killing ability against Ramos cells in the presence of Rituximab at a 5: 1 and 2.5: 1 effector: target cell ratio. Indicated.
  • the CD16V-28OX40LZ CAR, the third generation CAR containing the OX40 ligand showed the strongest killing ability compared to other CARs (FIG. 1C).
  • CD16V-OX40LZ CAR was constructed to evaluate cell killing ability. Transduction of CD16V-OX40LZ into NK92MI cells using a lentiviral vector was not expressed (FIG. 1D) and showed no killing ability against Ramos cells even in the presence of rituximab (FIG. 1E).
  • the OX40 ligand is a type II protein and the CD3 ⁇ linked to the OX40 ligand is a type I protein, thus changing the order of the two domains so that CD3 ⁇ is located at the N-terminus and the OX40 ligand is located at the C-terminus.
  • the inventors note that the CD16V-ZOX40L into NK92MI cells using a lentiviral vector containing an MSCV promoter, it was confirmed that the CD16V-OX40LZ was effectively expressed well (FIG. 1F).
  • CD16V-BBZ CAR (2nd generation) and CD16V-BBOX40LZ CAR (3rd generation) were expressed at high levels in NK92MI cells (FIG. 2A).
  • CD16V-Z, CD16V-BBZ, and CD16V-BBOX40LZ all showed low killing ability against Ramos cells when Rituximab was absent in the in vitro cell killing test.
  • CD16V-BBOX40LZ with OX40 ligand in proportion showed strong cell killing ability (FIG. 2B).
  • NK92MI cells expressing a CD16V-28OX40LZ CAR and NK92MI cells expressing a third generation CAR having a different intracellular signaling domain were all found to be expressed at high levels in NK92MI cells (FIG. 3A).
  • FIG. 3B Comparison of NK cell activation efficacy of various third generation chimeric antigen receptors (CAR) according to the present invention is summarized in FIG. 3B.
  • CAR third generation chimeric antigen receptors
  • FIG. 3B shows that all of the above CARs showed low cell killing ability against Ramos cells when rituximab was not present and the degree of cell killing was similar, but CD16V-28OX40LZ when rituximab was present.
  • CAR, CD16V-28OX40Z CAR and CD16V-28BBZ CAR all showed high cell killing ability against Ramos cells, among which CD16V-28OX40LZ CAR including OX40 ligand showed the best cell killing ability (FIG. 3B).
  • hinge sequence and composition between antigen-specific receptors and cell membranes are important.
  • the sequence and configuration of the hinge may need to be designed differently depending on the target molecule.
  • the CARs used in the experiments described above all used fragments of CD8 ⁇ as spacer domains.
  • the fragments of CD28 were used in the hinge, the cell killing ability of these CARs was evaluated and summarized in FIG. 4.
  • We have made a lentiviral vector comprising the extracellular domain of CD16V and the hinge of CD28.
  • the hinge, transmembrane and intracellular signaling domains derived from CD28 were linked to signaling modules of CD134 (OX40), CD137 (4-1BB), or OX40 ligand (CD252) in the 3rd generation CARs prepared.
  • the third generation CARs thus prepared were expressed in NK92MI cells using lentiviral vectors.
  • Transduced NK92MI cells were found to express high levels of each of the above 3rd generation CAR CD16V-28 (H) BBZ CAR, CD16V-28 (H) OX40Z CAR or CD16V-28 (H) OX40LZ CAR). 4A).
  • CD16V-28 (H) BBZ CAR, CD16V-28 (H) OX40Z CAR and CD16V-28 (H) OX40LZ CAR did not show cell killing ability when Rituximab was absent, but Ritux In the presence of simab, both CD16V-28 (H) BBZ CAR, CD16V-28 (H) OX40Z CAR and CD16V-28 (H) OX40LZ CAR showed strong cell killing ability. Among them, the CD16V-28 (H) OX40LZ CAR containing the OX40 ligand showed the highest cell killing ability.
  • Example 3 comprising OX40 ligand (CD252) NKG2D chimera Antigen receptor ( NKG2D Expressing -CAR) NK92MI Cytotoxicity Evaluation of Cells against Human Breast Cancer Cells and Lung Cancer Cells
  • NKG2D-Z CAR (1st generation), NKG2D-28Z CAR, NKG2D-BBZ CAR and NKG2D-OX40Z CAR (2nd generation) expressing NK-92MI cells were tested for MCF7 breast cancer cells. It was cytotoxic.
  • NKG2D-Z generation 1 CAR incorporation of CD134 (OX-40) or CD137 (4-1BB) costimulatory domains with CD3 ⁇ in the backbone of NKG2D-Z generation 1 CAR was higher than the cytotoxicity of NKG2D-Z CAR (generation 1) There was no toxicity.
  • Tumor cell killing ability by NK-92MI cells, including NKG2D-28Z CAR (2nd generation) was superior to NK-92MI cells expressing the positive control NKG2D-Z CAR (1st generation) (FIG. 5B).
  • CD137 4-1BB
  • OX40 CD134
  • TNFR tumor necrosis factor
  • NKG2D CAR (3rd generation) constructs were expressed in NK92MI cells using lentiviral vectors.
  • Transduced NK92MI cells efficiently expressed various NKG2D containing third generation CAR (FIG. 6A).
  • NK92MI cells expressing various NKG2D CARs (3rd generation) effectively lysed MCF7 cells in vitro.
  • NKG2D-28OX40Z CAR and NKG2D-28BBZ CAR which contained CD134 or CD137 costimulatory domains in the backbone of NKG2D-28Z CAR (2nd generation), did not show higher cytotoxicity than NKG2D-Z CAR (1st generation) (FIG. 6B).
  • NKG2D-28 (H) OX40LZ CAR (3rd generation) was fused by fusing the signal transduction region of the OX-40 ligand to the backbone of the NKG2D-28Z CAR (2nd generation).
  • Specific cytotoxicity induced by NKG2D-28 (H) OX40LZ CAR (3rd generation) on MCF7 cells in tumor cell death assay was significantly enhanced compared to NKG2D-Z CAR (1st generation), but NKG2D-28Z CAR (2nd generation) ) (FIG. 7B).
  • NKG2D ligand-positive tumor cells H1299 and H1944
  • NK92MI cells containing -28 (H) OX40L CAR (3rd generation) were NKG2D-AAA It was used as a target of NK92MI cells containing -28 (H) OX40L CAR (3rd generation).
  • Transformed NK92MI cells efficiently expressed NKG2D-Z CAR (1st generation) or NKG2D-AAA-28 (H) OX40L CAR (3rd generation) (FIG. 8A).
  • NKG2D ligand expression in H1299 and H1944 cells was evaluated.
  • NKG2D ligand expression was detected in H1299 and H1944 cells by flow cytometry (FIG. 8B).
  • NK92MI cells containing NKG2D-AAA-28 (H) OX40L CAR (3rd generation) were targeted to express NKG2D ligand in vitro than control NK92MI cells and NKG2D-Z CAR (1st generation) expressing NK92MI cells.
  • the cells could be lysed more efficiently.
  • Specificity was clearly confirmed by lysis of H1299 and H1944 cells by NK92MI cells expressing a control vector that does not express NKG2D.

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